The Diheme Cytochrome c4 from Vibrio cholerae Is ... - ACS Publications

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Biochemistry 2010, 49, 7494–7503 DOI: 10.1021/bi1004574

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The Diheme Cytochrome c4 from Vibrio cholerae Is a Natural Electron Donor to the Respiratory cbb3 Oxygen Reductase† Hsin-Yang Chang,‡,^ Young Ahn,‡,^ Laura A. Pace,‡ Myat T. Lin,§ Yun-Hui Lin, and Robert B. Gennis*,‡,§,

Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, §Center for Biophysics and Computational Biology, University of Illinois, Urbana, Illinois 61801, and Department of Chemistry, University of Illinois, Urbana, Illinois 61801. ^ These authors contributed equally to this work. )

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Received March 26, 2010; Revised Manuscript Received July 31, 2010 ABSTRACT: The respiratory chain of Vibrio cholerae contains three bd-type quinol oxygen reductases as well as

one cbb3 oxygen reductase. The cbb3 oxygen reductase has been previously isolated and characterized; however, the natural mobile electron donor(s) that shuttles electrons between the bc1 complex and the cbb3 oxygen reductase is not known. The most likely candidates are the diheme cytochrome c4 and monoheme cytochrome c5, which have been previously shown to be present in the periplasm of aerobically grown cultures of V. cholerae. Both cytochromes c4 and c5 from V. cholerae have been cloned and expressed heterologously in Escherichia coli. It is shown that reduced cytochrome c4 is a substrate for the purified cbb3 oxygen reductase and can support steady state oxygen reductase activity of at least 300 e-1/s. In contrast, reduced cytochrome c5 is not a good substrate for the cbb3 oxygen reductase. Surprisingly, the dependence of the oxygen reductase activity on the concentration of cytochrome c4 does not exhibit saturation. Global spectroscopic analysis of the time course of the oxidation of cytochrome c4 indicates that the apparent lack of saturation is due to the strong dependence of KM and Vmax on the concentration of oxidized cytochrome c4. Whether this is an artifact of the in vitro assay or has physiological significance remains unknown. Cyclic voltammetry was used to determine that the midpoint potentials of the two hemes in cytochrome c4 are 240 and 340 mV (vs standard hydrogen electrode), similar to the electrochemical properties of other c4-type cytochromes. Genomic analysis shows a strong correlation between the presence of a c4-type cytochrome and a cbb3 oxygen reductase within the β- and γ-proteobacterial clades, suggesting that cytochrome c4 is the likely natural electron donor to the cbb3 oxygen reductases within these organisms. These would include the β-proteobacteria Neisseria meningitidis and Neisseria gonnorhoeae, in which the cbb3 oxygen reductases are the only terminal oxidases in their respiratory chains, and the γ-proteobacterium Pseudomonas stutzeri.

The genome of Vibrio cholerae encodes four respiratory oxygen reductases (1). There are three bd-type oxygen reductases that use ubiquinol as the natural electron donor (2) and one cbb3-type heme-copper oxygen reductase (3), which is presumed to use a cytochrome c as its natural electron donor. The motivation for this work was to identify the natural electron donor for the cbb3-type heme-copper oxygen reductase. The cbb3-type oxygen reductases are members of the hemecopper superfamily that includes enzymes that perform oxygen reductase chemistry or nitric oxide reduction chemistry (4-7). The vast majority of currently identified oxygen reductases within the heme-copper superfamily are classified as being in one of three families, the A, B, and C families (6, 7). The cbb3-type oxygen reductases (7-11) are members of the C family and represent more than 20% of the sequences of heme-copper oxygen reductases from the currently known genome sequences. In the past decade, cbb3-type oxygen reductases have been purified and characterized from several bacterial species, including †

This work was supported by National Institutes of Health Grant HL16101 to R.B.G. *To whom correspondence should be addressed: Department of Biochemistry, A320 CLSL, MC-712, 600 S. Goodwin Ave., University of Illinois, Urbana, IL 61801. Telephone: (217) 333-9075. Fax: (217) 2443186. E-mail: [email protected]. pubs.acs.org/Biochemistry

Published on Web 08/17/2010

Rhodobacter sphaeroides (5, 12-17), Paracoccus denitrificans (18), Rhodobacter capsulatus (19-23), V. cholerae (3, 24), Bradyrhizobium japonicum (25-31), Rhodothermus marinus (32), Pseudomonas stutzeri (33-37), Helicobacter pylori (38, 39), and Sulfurihydrogenibium azorense (10). It has been demonstrated by mass spectrometry that the cbb3-type oxygen reductases have the signature histidine-tyrosine cross-linked cofactor within their active site, but that the tyrosine comes from a different transmembrane span than for the A family and B family heme-copper oxygen reductases (15, 24). The physiological role of the cbb3-type oxygen reductases is generally defined by a high affinity for O2, allowing these enzymes to function at lower oxygen concentrations. Therefore, these enzymes are often expressed under microaerophilic growth conditions (9). These enzymes are of particular interest because they are present in a number of pathogenic species and are proposed to be important for virulence by facilitating growth under conditions of low oxygen (40). The cbb3-type enzymes are the only oxygen reductases in H. pylori (41), Neisseria gonorrheae (42), and Neisseria meningitidis (43) and are, therefore, potential drug targets for these significant human pathogens. To understand the physiological roles of the cbb3-type oxygen reductases, it is important to know the natural electron donor(s). In most cases, the natural electron donor is not known. r 2010 American Chemical Society

Article One exception is the cbb3-type oxygen reductase from H. pylori, where the natural electron donor has been shown to be cytochrome c553 (39). When the electron donors are not known, the purified enzymes are usually assayed using the artificial electron donor TMPD1 (reduced by ascorbate). In a few instances, reduced horse heart cytochrome c can function as an electron donor (25), though often these assays are also performed in the presence of TMPD. The proteomes of V. cholerae strains have previously been analyzed for the presence of c-type cytochrome binding domains by searching for the CXXCH canonical sequence (44). A total of 48 CXXCH motifs were identified in 31 proteins, of which 14 have N-terminal signal sequences, indicating these are cytochrome c’s and are components of the bacterial envelope. Under aerobic growth conditions, six major c-type cytochromes are visualized by SDS-PAGE analysis using a stain for covalently bound heme (44). Two of these six cytochrome c’s have been identified as the CcoO and CcoP subunits of the cbb3-type oxygen reductase (44). A third heme-containing band was tentatively identified as the YecK subunit of biotin sulfoxide reductase (44). It is likely that one of the heme-staining bands corresponds to the PetC gene, encoding the cytochrome c1 component of the bc1 complex, but this remains to be seen. The best candidates for the remaining two heme-staining bands in aerobically grown V. cholerae are the cycA and cycB gene products, corresponding to cytochrome c4 and cytochrome c5, respectively. Each of these cytochrome c’s is predicted to have a cleaved N-terminal signal sequence and to be located within the periplasm. The predicted molecular masses of the mature cytochromes c4 and c5 (19.8 and 11.5 kDa, respectively) also make these reasonable candidates for two of the heme-staining bands from aerobically grown V. cholerae (44). The V. cholerae genome also encodes two additional c-type cytochromes of unknown function that are predicted to be periplasmic (44). Their predicted molecular masses (9.1 and 8.5 kDa) do not match those of the major heme-staining proteins reported for aerobically grown V. cholerae (44). It is likely, therefore, that cytochrome c4 and/or cytochrome c5 might be an electron donor to the cbb3-type oxygen reductase and responsible for shuttling electrons between the bc1 complex and the heme-copper oxygen reductase. For this reason, the genes encoding the diheme cytochrome c4 and monoheme cytochrome c5 were each cloned and expressed heterologously in Escherichia coli, and the proteins were purified. It is shown that cytochrome c5 does not function as an electron donor to the purified cbb3-type oxygen reductase from V. cholerae, whereas cytochrome c4 can support oxygen reductase activity at a rate of at least 300 e-1/s at 25 C. MATERIALS AND METHODS Construction of Expression Plasmids. A heterologous E. coli expression system (45) was used to obtain recombinant cytochromes c4 and c5 from V. cholerae. Following the procedure successfully employed previously for the expression of cytochrome c552 from Thermus thermophilus (46), the DNA fragments encoding the predicted sequence of the mature cytochromes c4 and c5 were fused to a DNA fragment encoding the signal peptide (MKISIYATLAALSLALPAGA) of cytochrome c550 from Thiobacillus versutus (47). The genes of the mature cytochrome c’s were 1 Abbreviations: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PEG, pyrolytic graphite edge; TMPD, N,N,N0 ,N0 tetramethyl-p-phenylenediamine; TMBZ, 3,30 ,5,50 -tetramethylbenzidine.

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obtained by polymerase chain reaction (PCR) from genomic DNA. The following oligonucleotides were used: 50 -CAAGGCGCCCAGGCCCAAGGTAGTATCGAAG-30 (forward primer) and 50 -AAGGATCCCTAGTGTAGGCCACCTAC-30 (reverse primer) for cycA and 50 -CAAGGCGCCCAGGCTCTAACTGAAGCCGATA-30 (forward primer) and 50 -AAGGATCCTTACAGGCCTGCGATCATA-30 (reverse primer) for cycB. The primers were designed for the introduction of a 50 NarI site and a 30 BamHI site to create a full-length chimeric T. versutus/V. cholerae cycA or cycB gene and cloned into pET17b. In addition, the 24th residue of cytochrome c5 was changed from Leu to Gln to enhance the recognition by the E. coli signal peptidase in cleaving the new cutting site. Both cytochromes c4 and c5 are predicted to have cleaved signal peptides and to be soluble, periplasmic proteins. The predicted cleavage sites are located after Ala36 and Ala23 for cytochromes c4 and c5, respectively. This hybrid construct was then cloned into commercial expression vector pET17b. Cell Growth and Enzyme Expression and Purification. Conditions of cell growth and enzyme expression were similar to those previously reported (46). The recombinant expression plasmids were utilized in a strain that also contained plasmid pEC86, which expresses maturation genes for cytochrome c in E. coli (ccmABCDEFGH) (27). The two plasmids were cotransformed into E. coli BL21(DE3) competent cells which were then streaked on a Luria-Bertani (LB) agar plate containing 50 μg/mL ampicillin and 30 μg/mL chloramphenicol (27, 46). The cells were grown in 1 L of culture medium in a 2.8 L Fernbach flask to an A600 of 0.8-1, and then expression of the cytochrome c was induced with 1 mM IPTG. After 7 h, the cells were harvested by centrifugation at 7000g for 15 min. Generally, ∼10 g of cell paste was collected from each liter of growth medium. The cell pellet was a reddish-brown color, indicating the overexpression of cytochrome c. Protein Purification. Each of the recombinant cytochrome c’s was purified by column chromatography, using procedures similar to those used for the purification of recombinant cytochrome c552 from Th. thermophilus (46). Briefly, the collected cell pellet was homogenized in 25 mM Tris-HCl (pH 7.5), 8 mM MgSO4, DNase I, 0.1% Triton X-100, and protease inhibitor cocktail. The suspended cell mixture was passed through a microfluidizer three times at a pressure of 20000 psi. The cell debris was spun down at 8000 rpm for 30 min at 4 C. The supernatant was loaded onto a 5 cm 20 cm CM-52 cellulose column (Whatman) and eluted with a gradient from 0 to 1 M NaCl in 25 mM Tris-HCl (pH 7.5) buffer. Fractions that were reddish-brown were collected and concentrated using concentrators (Amicon) with YM-10 membranes. The concentrated protein was loaded onto a 2.6 cm 100 cm gel filtration column (Sephacryl S-100 High Resolution, GE Healthcare) equilibrated with 100 mM potassium phosphate buffer (pH 7.0) at a flow rate of 0.4 mL/min. The protein was collected and dialyzed overnight against 25 mM Tris-HCl and then loaded onto the bufferequilibrated 1.6 cm 20 cm DEAE-5PW column (Toso-HaaS). The column was eluted using a gradient from 0 to 1 M NaCl in 25 mM Tris-HCl (pH 7.5) buffer. The eluted protein was dialyzed and concentrated again as described above, flash-frozen in liquid nitrogen, and stored at -80 C. The final yields of the purified cytochromes c4 and c5 were ∼15 and ∼5 mg/L of cell culture, respectively. SDS-PAGE Analysis. The purified c-type cytochromes were analyzed using SDS-PAGE. Protein was visualized using

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FIGURE 1: SDS-PAGE of the isolated recombinant cytochromes c4 and c5. (A) A 15% SDS gel stained with Coomassie blue. (B) Identical gel stained for heme. The lanes from left to right contain cytochrome c5 (lane 1), cytochrome c4 (lane 2), and molecular weight standards (lane 3).

Coomassie Blue, and heme staining (48) was used to identify proteins containing covalently attached heme c. Precast 15% SDS-PAGE gels from ISC BioExpress were used. After electrophoreses, the gels were then incubated in 15 mL of 6.3 mM 3,30 ,5,50 -tetramethylbenzidine (TMBZ from Sigma) and 35 mL of 0.25 M sodium acetate (pH 5.0) for 1 h. The gels were then stained for heme via addition of H2O2 to a final concentration of 30 mM. Spectrophotometric Measurements. Spectra of the isolated cytochromes were acquired with a Shimadzu UV-vis 2101PC spectrophotometer. The enzyme sample (3-17 μM) was examined in 25 mM Tris-HCl buffer at pH 7.5. The enzymes were oxidized with 2 μL of 1 mM Fe(CN)6 and reduced with a small amount of solid dithionite, both obtained from Sigma. Spectra were scanned from 375 to 800 nm. The concentrations of the cytochrome c’s were estimated from the spectra of the reduced cytochromes using extinction coefficients of 40 mM-1 cm-1 at 553 nm for the diheme cytochrome c4 (49, 50) and 20 mM-1 cm-1 at 555 nm for the monoheme cytochrome c5 (51). Cyclic Voltammetry. Reduction potentials were measured using protein film voltammetry (52, 53). The cytochrome c4 was applied directly to a freshly polished pyrolytic graphite edge (PGE) electrode surface and then placed into solution in a thermostated all-glass cell encased in a Faraday cage. Because oxygen does not interfere with measurements in the range of 100-500 mV [vs standard hydrogen electrode (SHE)], measurements were performed aerobically. Analogue-scan cyclic voltammetry was performed using a Bioanalytical Systems (West Lafayette, IN) CV-27 voltammograph with an in-house amplifier, and results were recorded via an in-house program. Data were analyzed using Fourier transformation and Origin 7.5 (OriginLab Corp., Northampton, MA). The solution pH value was controlled by using 10 mM HEPES buffer containing 2 M NaCl (pH 7.0). Purification of the cbb3-Type Oxidase from V. cholerae. The cbb3-type oxidase from V. cholerae was purified as previously described (3).

Steady State Kinetics Using an Oxygen Electrode. Cytochrome c oxidase activity was measured polarographically at 25 C using a YSI model 53 oxygen meter. The standard reaction mixture contained 1.8 mL of 50 mM sodium phosphate (pH 6.5), 50 mM NaCl, 0.05% dodecyl maltoside, 10 mM sodium ascorbate, and the cytochrome c to be tested. The cytochrome c4 and cytochrome c5 concentrations were varied in the range of 1100 μM. The O2 consumption reaction was initiated by the addition of the oxidase to a final concentration of 50 nM. The dependence of activity on ionic strength was measured by varying the concentration of NaCl from 10 to 150 mM using 20 μM cytochrome c4 reduced by 10 mM sodium ascorbate in 10 mM sodium phosphate (pH 6.5) buffer and 0.05% dodecyl maltoside. Steady State Kinetics Assessed by Stopped-Flow Spectrophotometry. To prepare the prereduced cytochrome c4, the sample was reduced with sodium dithionite and the excess dithionite was removed by gel filtration. The steady state kinetics between the cbb3 oxidase from V. cholerae and the prereduced cytochrome c4 was monitored at 25 C with Applied Photophysics SX-17MV stopped-flow spectrometer as follows. One syringe of the stopped-flow apparatus was filled with 400 nM cbb3 oxygen reductase in 50 mM sodium phosphate (pH 6.5), 150 mM NaCl, and 0.05% dodecyl maltoside, while the prereduced cytochrome c4 in the same buffer was loaded into the other syringe. The concentration of cytochrome c4 was varied from ∼1 to 60 μM for different measurements. After the sample had been mixed, the reaction was followed spectrometrically from 400 to 700 nm for 50-1000 s using a photodiode array. Using a least-squares fitting method (54), the spectrum at each time point was deconvoluted into the component spectra of reduced and oxidized cytochrome c4, which had been collected separately when cytochrome c4 was mixed with either sodium dithionite or potassium ferricyanide. The concentrations of the reduced and oxidized cytochrome c4 at each time point were then used to calculate the reaction rate at each point. By using different starting concentrations of cytochrome c4 in different measurements, the reaction rates at various combinations of reduced and oxidized cytochrome c4 concentrations were obtained.

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FIGURE 2: Absorption spectra of fully reduced (;) and fully oxidized ( 3 3 3 ) V. cholerae (A) cytochrome c4 (3 μM) and (B) cytochrome c5 (7 μM). The insets show the R and β bands of the fully reduced cytochrome c4 (10 μM) and cytochrome c5 (17 μM). All the absorption spectra were recorded in 25 mM Tris-HCl (pH 7.5) at 25 C.

The reaction rates were plotted versus reduced cytochrome c4 concentrations at a fixed oxidized cytochrome c4 concentration, and the KM and Vmax for each plot were obtained by nonlinear least-squares fitting to the standard Michaelis-Menten kinetics model. All data processing and analyses were performed with Mathematica. RESULTS Expression and Purification of Recombinant Cytochrome c4 and Cytochrome c5. Transformants were found to

express the cytochrome c’s upon induction (46), and the recombinant proteins were isolated as described in Materials and Methods. The SDS-PAGE analysis of the recombinant cytochromes is shown in Figure 1. After being stained with Coomassie Blue (Figure 1A), the purified cytochrome c4 has a single band near 21-22 kDa that contains two covalently bound hemes (Figure 1B). The expected molecular weight of the mature cytochrome c4 is 19791 (187 amino acid residues) with 1233 (two-heme c moiety). Cytochrome c5 has a single heme-staining

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FIGURE 3: Cyclic voltammetry of the recombinant cytochrome c4 that was applied to a freshly polished pyrolytic PGE electrode. The solution contained 10 mM HEPES buffer and 2 M NaCl (pH 7.0). The following values were obtained (vs SHE): Epc1 = 0.26 V, Epc2 = 0.35 V, Epa1 = 0.23 V, Epa2 = 0.34 V, and E1/2 = (Epc þ Epa)/2 = 0.24 and 0.34 V.

component running near the expected molecular weight of 11480, but there is a major protein contaminant that does not contain heme. Because cytochrome c5 does not function as a reductant for the cbb3-type oxidase (see below), no further effort was made to improve the purification. UV-Vis Spectroscopy. Figure 2 shows the spectra of the reduced and oxidized forms of cytochromes c4 and c5. The spectra of each are consistent with previously published reports of homologues from other organisms (49-51). In the reduced form, the absorption spectrum of cytochrome c4 has three maxima at 553, 523, and 417 nm, corresponding to the R, β, and γ (Soret) bands, respectively (Figure 2A). The maxima of the oxidized form of cytochrome c4 are at 528 and 409 nm. The spectra show two diagnostic features shared by all cytochrome c4’s: (1) a split R-band with maxima at 553 and 549 nm and (2) a low ratio of the amplitude of the R band to the β band (A553/A523 = 1.05). Reduced cytochrome c5 has maxima at 554.8, 524.6, and 419 nm, corresponding to the R, β, and γ (Soret) bands, respectively (Figure 2B). The R/β ratio A555/A525 = 1.5. The spectrum of the oxidized cytochrome c5 has two peaks at 526 and 413 nm. These features are consistent with previous reports of cytochrome c5’s (51). Electrochemistry. Cytochrome c4 was found to be amenable to direct electrochemical characterization when adhered to a carbon electrode. The midpoint potentials of the two heme components of the recombinant cytochrome c4 were measured by cyclic voltammetry (Figure 3). Four well-defined redox peaks are observed, and the Eom values of the two heme groups were determined to be approximately 240 and 340 mV (vs SHE). Rates of Oxygen Reduction by the V. cholerae cbb3-Type Oxidase Using Cytochromes c4 and c5. Figure 4A compares the rates of oxygen reduction, measured with an oxygen electrode, by the V. cholerae cbb3-type oxidase using ascorbate and either (i) horse heart cytochrome c, (ii) recombinant cytochrome c4, or (iii) recombinant cytochrome c5. These assays were performed in the absence of the mediator TMPD, which can itself function as a substrate for the cbb3-type oxidase. The data (Figure 4A) show that neither the horse heart cytochrome c nor

Chang et al. cytochrome c5 is effective as a substrate, whereas cytochrome c4 is capable of supporting turnover at rates of >300 e-1/s at 25 C. This turnover is comparable to what is observed with 0.5 mM TMPD in the presence of ascorbate to maintain the TMPD reduced (not shown). The turnover using recombinant cytochrome c5 is more than a factor of 10 slower (7%) than that observed with cytochrome c4, and horse heart cytochrome c is also a poor substrate (8%). Although the purity of recombinamt cytochrome c5 was not high, it is unlikely that the contaminants are inhibitory. The oxidase activity with cytochrome c4 is rapidly stopped upon addition of 25 μM cyanide (Figure 4B), showing that the oxidase activity is catalyzed by the cbb3-type oxygen reductase. The data in Figure 4A do not indicate saturation even at concentrations of cytochrome c4 as high as 100 μM. Activity was compared using 50 nM oxidase and 20 μM cytochrome c4, varying the concentration of NaCl from 1 to 150 mM (Figure 4C). These data indicate that the apparent binding of cytochrome c4 to the oxidase is not sensitive to ionic strength and that the lack of saturation of activity as a function of the concentration of cytochrome c4 is not due to weak binding due to the selected ionic strength. It was determined, however, that under the conditions used for the assays with the oxygen electrode, the ascorbate does not keep the cytochrome c4 fully reduced. Stopped-Flow Spectrophotometry. To improve our understanding of the lack of saturation in the steady state kinetics measurements using the oxygen electrode, steady state activity was also measured by stopped-flow spectrophotometry, mixing prereduced cytochrome c4 with a solution containing the cbb3type oxidase. The oxidation of cytochrome c4 was monitored by recording the change in the absorption spectrum from 400 to 700 nm using a photodiode array. The initial concentration of cytochrome c4 was varied from 1 to 60 μM, and spectra were recorded as a function of time for each initial condition. The spectra were deconvoluted to quantify the concentration of oxidized and reduced cytochrome c4 in solution as a function of time. From this large matrix of data, the rate of oxidation of cytochrome c4 could be determined for a wide range of concentrations of both the reduced and oxidized forms of cytochrome c4. These data were plotted to show how the rate of oxidation varies as a function of reduced cytochrome c4 concentration in the presence of a specific concentration of oxidized cytochrome c4 (Figure 5A-E). If the oxidized cytochrome c4 is a competitive inhibitor for the reduced cytochrome c4, one would expect that the data would show a constant Vmax and increasing KM as the concentration of oxidized cytochrome c4 increases. However, this is not the case, and both Vmax and KM increase as the concentration of oxidized cytochrome c4 increases (Figure 5F). One complication is that the spectral deconvolution with the least-squares method is not satisfactory in the case of cytochrome c4 because cytochrome c4 contains two hemes that have slightly different spectra. The assumption that either both are oxidized or both are reduced is not realistic. Attempts were not made to analyze the data with more complex models. Despite this, our analysis indicates the following significant points. (1) Oxidized cytochrome c4 has a strong effect on the activity between the cbb3-type oxidase and reduced cytochrome c4. The apparent KM increases in the presence of relatively small amounts of oxidized cytochrome c4 (Figure 5F). If the concentration of oxidized cytochrome c4 is greater than 2.5 μM, the KM of reduced cytochrome c4 is too large to obtain a reasonably accurate fitting to the Michaelis-Menten model. (2) During the manipulation of the prereduced cytochrome c4 to load the stopped-flow syringe, a significant amount of

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FIGURE 4: Steady state oxidase activity of the cbb3 oxygen reductase from V. cholerae. (A) Dependence on the concentration of cytochrome c4 (9), cytochrome c5 (O), and horse heart cytochrome c (2). The reaction mixture contained 50 mM sodium phosphate (pH 6.5), 50 mM NaCl, 0.05% dodecyl maltoside, 10 mM sodium ascorbate, and the indicated amount of the cytochrome c, in a total volume of 1.8 mL. The O2 consumption reaction was initiated by the addition of 50 nM oxidase. (B) Sensitivity of the oxidase activity to the addition of 25 μM cyanide. (C) Dependence of the oxidase activity on the concentration NaCl, measured with 50 nM oxidase and 20 μM cytochrome c4, in the presence of 10 mM sodium ascorbate, 10 mM sodium phosphate (pH 6.5) buffer, and 0.05% dodecyl maltoside.

autoxidation occurs. Hence, none of the data represent a starting condition under which cytochrome c4 is actually fully reduced. (3) Because of the spectroscopic complications of having two different heme components in cytochrome c4, data fits are not sufficient to extrapolate to obtain true values for either Vmax or KM. Phenomenologically, as the concentration of oxidized cytochrome c4 increases, both the apparent Vmax and KM increase. The inhibitory influence of oxidized cytochrome c4 and the increasing concentration of oxidized cytochrome c4 that accumulates as the reaction proceeds explain the lack of saturation under conditions used for the oxygen electrode assays (Figure 4A).

DISCUSSION Although initially thought to be confined to proteobacteria (7, 11), the cbb3 oxygen reductases are widely distributed among bacterial phyla (5, 10). Although there are exceptions, most archaea lack c-type cytochromes (55), and no example of a cbb3type oxidase in an archaea has been reported. Nearly all studies have focused on the role of the C family (cbb3) oxygen reductases in the aerobic respiratory chains of proteobacteria (8), exceptions being the cbb3-type oxidases from Rh. marinus (yet to be confirmed in the genome sequence) (32) and from S. azorense (10).

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FIGURE 5: Rates of oxidation of reduced cytochrome c4 by the cbb3 oxygen reductase from V. cholerae at different oxidized cytochrome c4 concentrations determined by stopped-flow spectroscopy and least-squares fitting. The substrate S refers to reduced (not total) cytochrome c4. The oxidized cytochrome c4 concentrations were (A) 1.5, (B) 2.0, (C) 2.5, (D) 3.0, and (E) 7.5 μM. The curves show the nonlinear least-squares fitting of the points to Michaelis-Menten kinetics. The KM and Vmax values for 1.5, 2.0, and 2.5 μM oxidized cytochrome c4 are plotted in panel F.

In most cases, the natural electron donors for these C family oxygen reductases are not known. This work has identified the diheme cytochrome c4 as a natural electron donor to the cbb3type oxygen reductase from the γ-proteobacterium, V. cholerae. Cytochrome c4 is one of five cytochrome c’s encoded in the genome of V. cholerae that are predicted to be soluble, periplasmic proteins (44). Cytochromes c4 and c5 appear to be among the major cytochrome c’s expressed under aerobic growth conditions (44). Heterologous expression in E. coli was successful, generating soluble cytochromes c4 and c5. Cytochrome c4 from V. cholerae has spectroscopic and electrochemical properties similar to those of the cytochrome c4’s from other organisms. These include the high midpoint potentials (240 and 340 mV, vs SHE) of the two hemes, the split R band, and the low ratio of the peak heights of the R and β bands (50, 56, 57). Cytochrome c4 can clearly donate electrons to the cbb3-type oxidase, supporting steady state reduction of O2 at a rate of at least 300 e-1/s. Rates attained using either cytochrome c5 or horse heart cytochrome c were 10-20-fold lower under comparable conditions. The one caveat is that the steady state rate of

cytochrome c4 oxidase activity does not saturate and quite high concentrations (100 μM) are required to reach the turnover rate of 300 s-1. The lack of saturation (Figure 4A) is unusual and not expected. By comparison, the rate of steady state turnover of H. pylori cytochrome c553 by the H. pylori cbb3-type oxidase is 250 s-1, with a Km of 0.9 μM. The nearly linear dependence of oxygen reductase activity as a function of the concentration of cytochrome c4 (Figure 4A) can be qualitatively explained as being due to the influence of oxidized cytochrome c4 (39). There is a significant concentration of oxidized cytochrome c4 present at all times during the oxygen reductase assay, and the presence of ascorbate at a concentration as high as 100 mM does not alleviate this problem. The data do show that the true Vmax for the oxidation of cytochrome c4 must be at least 300 s-1, but the true Km cannot be measured using this assay. In the steady state assay, the velocity is measured in the presence of 100 μM reduced cytochrome c4 with ∼25 μM oxidized c4 also present. The stopped-flow experiments cannot be performed under these conditions, but the data show that the values of both Vmax and KM depend on the concentration of oxidized cytochrome c4, and

Article the stopped-flow data are consistent with the steady state kinetics. It is quite conceivable that the true Km is in the range of 1-10 μM, but further work will be needed to determine this. Because the oxidized and reduced forms of cytochrome c often have similar affinities for their redox partners, it is to be expected that oxidized cytochrome c4 would competitively inhibit the activity between reduced cytochrome c4 and the cbb3-type oxidase. However, product inhibition can explain only the increase in the KM but not the increase in Vmax that is observed as the concentration of the product increases (Figure 5). Whether this kinetic behavior reflects an in vitro artifact or an unusual form of allosteric regulation is not clear. This unusual behavior might be related to the fact that cytochrome c4 is a two-electron carrier. Cyclic voltammetry shows that the diheme cytochrome c4 exhibits two substantially different midpoint potentials, but the extent of cooperativity between the two hemes is not known. In principle, there could be two different binding sites on cytochrome c4 for the cbb3 oxygen reductase, each with a distinct KM and Vmax. In any event, further characterization of the oxidation kinetics of the diheme cytochrome c4 is needed to address this question. At this point, it is safe to conclude that cytochrome c4 is definitely a substrate for the cbb3-type oxidase whereas cytochrome c5 is a very poor substrate, at best, under in vitro conditions. Roles of Cytochrome c4 in Other Bacteria. Analysis of sequenced genomes demonstrates that the distribution of cytochrome c4 is limited, with the majority of homologues being found within the β- and γ-proteobacterial clades. Cytochrome c4 is also sporadically distributed within the R-proteobacterial clade as well as a few other bacterial phyla. Bacteria within the β- and γ-proteobacterial clades that contain a cbb3 oxygen reductase are very likely also to contain a cytochrome c4. Within these groups of bacteria, it is reasonable to assume that cytochrome c4 donates electrons to the cbb3 oxygen reductase. It is noted that the cbb3 oxygen reductases within the β- and γ-proteobacterial clades all contain the auxiliary subunit CcoP, a diheme cytochrome c that is the likely electron acceptor from cytochrome c4. A large number of the cbb3 oxygen reductases outside the β- and γ-proteobacterial clades are missing the CcoP subunit, and others have an alternative subunit, CcoR (10). Indeed, many organisms that are outside the β- and γ-proteobacterial clades that encode a cbb3 oxygen reductase do not contain cytochrome c4 and, therefore, must be using a different donor of electrons to this enzyme. Other γ-proteobacteria that contain cbb3 oxygen reductases include Azotobacter vinelandii (58), and P. stutzeri (33-37). Knockout mutants of both cytochromes c4 and c5 in A. vinlandii support a role for both of these cytochromes in aerobic respiration, though there are no data concerning a specific role with the cbb3 oxygen reductase. The cytochrome c4 from P. stutzeri has been extensively characterized (49, 59-62), but its physiological role has not yet been defined. Acidothiobacillus ferrooxidans is a γ-proteobacterium whose genome includes several cytochrome c4’s but not a cbb3-type oxidase (63-65). The cytochrome c4’s are strongly implicated in the aerobic respiratory chain (66-68). Ac. ferrooxidans is an obligate chemolithotropic bacterium that grows at acidic pH (pH 1.5-4) and uses either ferrous iron or reduced sulfur compounds as an electron source. The cyc1 gene encodes a diheme c4-type cytochrome c552 within an operon that includes an A family oxygen reductase as well as the blue copper protein rusticyanin (69). These respiratory components comprise the electron transport chain between the ferrous iron oxidase and O2. The cytochrome c4 forms a complex with rusticyanin that is


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likely the immediate donor of electrons to the A family oxygen reductase (68, 70). The structure of the Cyc1 cytochrome c4 has been determined, and the complex with rusticyanin has been modeled computationally (64, 70-72). Two additional genes encoding cytochrome c4’s, cycA1 and cycA2, are in operons encoding two separate bc1 complexes (66). It is suggested that when growing on reduced sulfur compounds, CycA2 shuttles electrons from the bc1 complex encoded by the petII operon to one or several terminal reductases (66). When growing on Fe2þ, CycA1 is proposed to be part of an aerobic respiratory chain that runs in the reverse direction to generate NADH: CycA f bc1 (petI) f Q f Complex I (66, 67). The β-proteobacterial clade includes N. meningitidis and N. gonorrheae, which are each human pathogens and are notable also for the fact that the cbb3 oxygen reductase is the only respiratory oxidase encoded in their genomes (42, 43). Knockout mutations have implicated cytochrome c4 along with cytochrome cx (a homologue of cytochrome c552 from Th. thermophilus) as being a donor of electrons to the cbb3 oxygen reductase in N. meningitidis (73). Cytochrome c5 is required for the transfer of electrons to the nitrite reductase in N. meningitidis (73). Knockout mutations have also indicated a role of cytochrome c4 along with cytochrome c5 as substrates of the cbb3 oxygen reductase in N. gonorrheae, but not cytochrome c2 (42). Interestingly, the CcoP subunit of the cbb3-type oxidase in N. gonorrheae has an extra cytochrome c domain that is homologous to cytochrome c5, and this domain is implicated in the transfer of electrons to the nitrite reductase that is tethered to the outer membrane (74). Outside the β- and γ-proteobacterial clades, there is limited information about the native electron donors for the cbb3 oxygen reductase. One exception is H. pylori, in which the monoheme cytochrome c553 (39) is the substrate for the cbb3 oxygen reductase (38). H. pylori is an ε-proteobacterium. Closely related ε-proteobacteria that contain cbb3 oxygen reductases and might also use homologues of cytochrome c553 as substrates are Campylobacter jejuni (41, 75) and Wolinella succinogenes (76, 77). The cbb3 oxygen reductases have been biochemically characterized from several R-proteobacteria: R. sphaeroides (5, 12-17), Pa. denitrificans (18), R. capsulatus (19-23), B. japonicum (25-31), and Rh. marinus (32). Using knockout mutants, it was concluded that both the A and C family oxygen reductases from R. sphaeroides can utilize either cytochrome c2 or membraneanchored cytochrome cy (21). CONCLUSION Cytochrome c4 has been shown to be the native electron donor for the C family (cbb3) oxygen reductase in V. cholerae. Within the γ- and β-protobacterial clades, there is a strong correlation for the coexistence of a C family oxygen reductase and a cytochrome c4, suggesting that in these organisms, it is likely that cytochrome c4 is a natural electron donor. This does not exclude other electron donors from also being substrates within these organisms. Outside the γ- and β-protobacterial clades, there is clear evidence that other cytochrome c’s are substrates for C family oxygen reductase, though in most cases, there are no data to identify these electron donors. ACKNOWLEDGMENT We thank Dr. SangMoon Lhee and Dr. Antony R. Crofts of the Department of Biochemistry, University of Illinois, for technical assistance.

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